Cystic fibrosis (CF) is the most common fatal autosomal recessive disease among Caucasians, with a frequency of ~1 in 2500 live births and is caused by recessive mutations in the cystic fibrosis transmembrane conductance regulatory (CFTR) gene which encodes a membrane chloride channel present in the epithelium of the lung and other organs. Morbidity and mortality in CF patients is due to lung disease characterized by inflammation, obstructive mucus and persistent infection. CF is currently inadequately treatable and CF patients inevitably succumb to respiratory failure at a median age of -32 years. Clearly novel therapies are needed and the desire to develop gene therapy to treat CF is high. However, despite over a decade of research, CF gene therapy has remained elusive. Chief among the obstacles to successful gene therapy are 1) inefficient gene transfer to the airway epithelium and submucosal glands, 2) toxicity of the gene transfer vectors and 3) the challenge of vector readministration. Therefore, the objective of this proposal is to specifically address each of these limitations. This will be accomplished by evaluating the safety and efficacy of a novel method of delivering helper-dependent adenoviral vectors (HDAd) into the lungs of nonhuman primates. HDAd are deleted of all viral coding sequences and are superior to first generation, E1-deleted, Ad vectors in terms of reduced toxicity and longer duration of transgene expression. We have recently developed novel methods to deliver HDAd into the lungs of nonhuman primates and have obtained exceedingly high and unprecedented levels of transduction of the airway epithelium and submucosal glands with negligible toxicity and minimal pulmonary inflammation. Therefore, we propose to build upon these encouraging preliminary results in this proposal. Specific Aim 1 is to optimize and assess the safety and efficacy of intratracheal aerosolization of HDAd to achieve uniform transduction throughout the lung. Specific Aim 2 is to optimize and assess the safety and efficacy of targeted lobar aerosolization of HDAd to achieve uniform transduction throughout the lung. Specific Aim 3 is to investigate the duration of transgene expression following pulmonary transduction and chronic toxicity. Specific Aim 4 is to investigate the safety and efficacy of various strategies of pulmonary readministration of HDAd. Successful completion of these Specific Aims will provide an important first step towards clinical CF gene therapy.